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What is RP-HPLC and why is it used for peptides?
The short answer
Reverse-phase high-performance liquid chromatography separates a mixture by how strongly each component sticks to a non-polar column while a changing solvent gradient washes it through. Peptides separate well by this mechanism, the equipment is widely available, and pharmacopoeial methods are built on it — which makes it the standard technique for peptide purity and quantification.
In detail
In practice the sample is injected, components travel at different speeds, and a UV detector records what emerges and when. The result is a chromatogram: peaks in time order, whose areas are integrated to give purity, and whose position identifies compounds against a standard.
Its strength is resolution and reproducibility. Its limit is that it identifies by behaviour, not by mass — which is why retention-time identity is presumptive and mass spectrometry is the confirmatory step.
Our full method, including column chemistry, gradient and wavelength, is published rather than described in general terms.
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